Electric field directed loading of microwell array

ABSTRACT

An apparatus includes a device substrate including an array of sensors. Each sensor of the array of sensors can include a electrode structure disposed at a surface of the device substrate. The apparatus further includes a wall structure overlying the surface of the device substrate and defining an array of wells at least partially corresponding with the array of sensors. The well structure including an electrode layer and an insulative layer.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation application of PCT Application No.PCT/US2012/058559, filed Oct. 3, 2012, which claims benefit of U.S.Provisional Application No. 61/542,611, filed Oct. 3, 2011, entitled“ELECTRIC FIELD DIRECTED LOADING OF MICROWELL ARRAY,” and which claimsbenefit of U.S. Provisional Application No. 61/550,193, filed Oct. 21,2011, entitled “ELECTRIC FIELD DIRECTED LOADING OF MICROWELL ARRAY,”which are incorporated herein by reference in their entirety.

FIELD OF THE DISCLOSURE

Embodiments of the present disclosure generally relate to the field ofmicrowell arrays. More specifically, embodiments of the presentdisclosure refer to loading of a microwell array with, for example,particles or microbeads using an electric field.

BACKGROUND

Electrochemical detection is attractive because it provides highsensitivity, small dimensions, low cost, fast response, andcompatibility with microfabrication technologies. These characteristicsled to the development of a variety of sensors based on amperometric,potentiometric, and impedimetric signals, and the assembly of sensorsinto an array format for chemical, biochemical, and cellularapplications. Typically, in such systems, analytes are distributed amongan array of confinement regions or microwells (also referred to hereinas “wells” or “reaction chambers”), and reagents are delivered to suchregions by a fluidics system that directs the flow of reagents through aflow cell containing the sensor array.

Some applications involve the distribution of nucleic acid moleculesattached to supports (e.g., particles or microbeads) in an array format.For example, several sequencing methods involve analysis of nucleic acidlibraries, where individual members of the libraries are attached toparticles that are distributed into an array of microwells. For suchapplications, increasing the number of microwells into which particles(or microbeads) are loaded can be desirable, because empty microwellsmay not provide useful information. The percentage of microwells thatreceive a particle or microbead can be referred to as the “loadingefficiency.” Alternatively, in sequencing applications the loadingefficiency can refer to the percentage of microwells in the arrayyielding a readable sequence. Poor loading efficiencies (e.g., loadingefficiencies less than 50%) increase the overall cost and effortassociated with a chemical/biological experiment.

Therefore, improved loading efficiencies in microwell arrays would bedesirable.

SUMMARY

An apparatus includes a device substrate including sensors. A wellstructure overlies the surface of the device substrate and defines anarray of wells at least partially corresponding with the sensors. Thewell structure includes an electrophoresis electrode layer and aninsulative layer.

Loading particles can be performed by providing a particle suspensioninto a flow cell of an apparatus that includes a wall structureincluding an electrode layer and an insulative layer and includes acounter electrode. A voltage source electrically coupled to theelectrode layer and the counter electrode can be activated to provide avoltage difference between the electrode layer and the counterelectrode, whereby the particles are motivated into wells of the arrayof wells.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure may be better understood, and its numerousfeatures and advantages made apparent to those skilled in the art byreferencing the accompanying drawings.

FIG. 1 is an illustration of a microwell and a sensor.

FIG. 2 is an illustration of an embodiment of a flow cell.

FIG. 3 is an illustration of an embodiment of a voltage source that isused in conjunction with a flow cell.

FIGS. 4-46 include illustrations of exemplary portions of exemplarysensor workpieces during stages of exemplary manufacturing processes.

FIG. 47 includes a block flow illustration of an exemplary loadingmethod.

The use of the same reference symbols in different drawings indicatessimilar or identical items.

DETAILED DESCRIPTION

The following detailed description refers to the accompanying drawingsthat illustrate exemplary embodiments consistent with this invention.Other embodiments are possible, and modifications can be made to theembodiments within the scope of the invention. Therefore, the detaileddescription is not meant to limit the invention.

It would be apparent to person of ordinary skill in the relevant artthat the present invention, as described below, can be implemented inmany different embodiments of hardware or the entities illustrated inthe figures. Thus, the operational behavior of embodiments of thepresent invention is described with the understanding that modificationsand variations of the embodiments are possible, given the level ofdetail presented herein.

FIG. 1 is an expanded and cross-sectional view of a flow cell 100 andshows a portion of a flow cell 106. A reagent flow 108 flows across asurface of a microwell array 102, in which the reagent flow 108 flowsover the open ends of the microwells. The microwell array 102 and asensor array 105 together may form an integrated unit forming a bottomwall (or floor) of flow cell 100. A reference electrode 104 may befluidly coupled to flow cell 106. Further, a flow cell cover 130encapsulates flow cell 106 to contain reagent flow 108 within a confinedregion. Exemplary flow cell structures and associated components can befound at U.S. Patent Application Publication No. 2010/0137143 (filed May29, 2009), which is incorporated by reference herein in its entirety.

FIG. 1 illustrates an expanded view of a microwell 101 and a sensor 114.The volume, shape, aspect ratio (such as base width-to-well depthratio), and other dimensional characteristics of the microwells may beselected based on the nature of the reaction taking place, as well asthe reagents, byproducts, or labeling techniques (if any) that areemployed. The sensor 114 can be a chemical field-effect transistor(chemFET) or an ion-sensitive field effect transistor (ISFET) (togetherreferred to as “FET”) with a floating gate 118 having a sensor plate 120optionally separated from the microwell interior by a passivation layer116. The sensor 114 can be responsive to (and generate an output signalrelated to) the amount of a charge 124 present on passivation layer 116opposite of the sensor plate 120 or on the sensor plate 120 itself.Changes in the charge 124 can cause changes in a current flowing betweena source 121 and a drain 122 of the FET. In turn, the FET can be useddirectly to provide a current-based output signal or indirectly withadditional circuitry to provide a voltage-based output signal.Reactants, wash solutions, and other reagents may move in and out of themicrowells by a diffusion mechanism 140.

In an embodiment, reactions carried out in the microwell 101 can beanalytical reactions to identify or determine characteristics orproperties of an analyte of interest. Such reactions can generatedirectly or indirectly byproducts that affect the amount of chargeadjacent to the sensor plate 120. If such byproducts are produced insmall amounts or rapidly decay or react with other constituents, thenmultiple copies of the same analyte may be analyzed in the microwell 101at the same time in order to increase the output signal ultimatelygenerated. In an embodiment, multiple copies of an analyte may beattached to a solid phase support 112, either before or after depositioninto the microwell 101. The solid phase support 112 may bemicroparticles, nanoparticles, beads, solid and porous comprising gels,or the like. For simplicity and ease of explanation, solid phase support112 is also referred herein as a particle or microbead. The particle cancarry a charge. In particular, polymer particles conjugated topolynucleotides can carry a charge. Alternatively, for a nucleic acidanalyte, multiple, connected copies may be made by rolling circleamplification (RCA), exponential RCA, or like techniques, to produce anamplicon without the need of a solid support.

Improved loading efficiency of the microwell array 102 is desirable. Theoverall cost and effort associated with the microwell experiment can beimproved with an increase in loading efficiency. In an embodiment, theloading efficiency of the microwell array 102 can be improved byintroducing an electric field within the flow cell 106 to direct thesolid phase support 112 into a microwell. The electric field directedloading of the microwell array 102 and structures to facilitate loadingare described in further detail below.

As illustrated in FIG. 1, a wall structure 110 defines the well 101. Thewall structure 110 can be formed of more than one layer and of more thanone material. In particular, the wall structure 110 includes anelectrophoresis electrode layer. A voltage, relative to a counterelectrode, can be applied to the electrophoresis electrode layer todrive particles, such as the solid phase support 112, into the well 101.

FIG. 2 is an illustration of a flow cell 200 according to an embodiment.The flow cell 200 includes a voltage source 210 with terminals 220 and230. The flow cell 200 also includes a counter electrode 240 that isdisposed along a surface of the flow cell cover 130. The counterelectrode 240 can be, for example, an electrically conductive ceramic,such as indium tin-oxide, titanium nitride, or a combination thereof, orcan be an electrically conductive polymeric material, such as conductiveepoxy or other coatings. The counter electrode 240 is electricallycoupled to the terminal 220, and the microwell array 102 is electricallycoupled to the terminal 230, according to an embodiment. The terminal230 is electrically coupled to the microwell array 102 via anelectrophoresis electrode layer implemented in the well wall structuresdefining the microwells in the microwell array 102. The nature of theelectrophoresis electrode in the well wall structures defining themicrowells is described in further detail below.

The voltage source 210 provides an electric field to the flow cell 106via the counter electrode 240 and the electrophoresis electrode inmicrowell array 102. The electric field directs a microbead (e.g., solidphase support 112 of FIG. 1) into a portion of the microwells in themicrowell array 102. That is, as microbeads enter the flow cell 106 in abuffer solution to be received by the microwells in the microwell array102, the electric field interacts with a charge on the microbeads, andthe charged microbeads are directed into the wells by electrophoresis.The microbeads can be captured by the microwells through chemical,mechanical or hydrostatic techniques. In an example, anelectrochemically-induced binding between a chemical property of themicrowell and a chemical property of the microbead can capture themicrobead in a microwell.

For instance, the microwells in the microwell array 102 can include agold layer, and the microbeads can be coated with streptavidin. As thestreptavidin-coated microbeads enter the electric field in the flow cell106 in a buffer solution, the streptavidin-coated microbeads can bedirected into the microwells by electrophoresis. Further, thestreptavidin-coated microbeads can be captured by the microwells throughbinding between the gold and streptavidin. An example of such bindingbetween gold and streptavidin can be found at Barbee et al., ElectricFiled Directed Assembly of High-Density Microbead Arrays, The RoyalSociety of Chemistry—Lab on a Chip, Sep. 15, 2009, Issue 22, at3268-3274, which is incorporated by reference herein in its entirety.

FIG. 3 is an illustration of the voltage source 210, according to anembodiment. The voltage source 210 includes a voltage supply 300 withterminals 310 and 320, a switch 330, and a pulse generator 350. Theterminals 310 and 320 can be negative and positive terminals of thevoltage supply 300, respectively. In an embodiment, the terminals 310and 320 are coupled to terminals 220 and 230 of FIG. 2, respectively; inturn, the voltage potential from the terminal 310 is transferred to thecounter electrode 240 of FIG. 2, and the voltage potential from theterminal 320 is transferred to the electrode in the microwell array 102of FIG. 2 via the switch 330. The terminals 310 and 320 of FIG. 3 can bethe positive or negative terminals of voltage supply 300. For ease ofexplanation, the terminals 310 and 320 are negative and positiveterminals of the voltage supply 300, respectively, and the terminals 310and 320 are coupled to the terminals 220 and 230 of FIG. 2, respective,for the description below.

In reference to FIG. 3, the pulse generator 350 and the switch 330 (withthe voltage source 300) provide electrical pulses to the flow cell 106of FIG. 2. In particular, based on the output of the pulse generator350, the switch 330 opens and closes to provide a pulsed output, andthus, an electric field is generated in the flow cell 106. The pulsegenerator 350 can be, for example, a function generator that delivers DCvoltage pulses (e.g., 3.0 V DC pulses) at a predetermined frequency andduty cycle (e.g., 1 Hz and a duty cycle of 10%). In another embodiment,a non-pulsed DC voltage can be delivered to the flow cell 106 togenerate the electric field. When the switch 330 of FIG. 3 is closed,the voltage potential from the voltage source 300 is transferred to themicrowell array 102 and the counter electrode 240 of FIG. 2 to generatethe electric field. Alternatively, an AC voltage, such as a biased ACvoltage, can be implemented in the embodiments described above.

Based on the electric field provided to the flow cell 106 of FIG. 2, themicrobeads in the flow cell 106 can be directed to empty microwells byelectrophoresis. Thus, as a result, the loading efficiency of themicrowell array 102 of FIG. 2 is increased.

The implementation of the electrode in each of the microwells inmicrowell array 102 is discussed. As discussed above, the electrodeassociated with the microwells is electrically coupled to a voltagepotential of the voltage source 300 of FIG. 3. In conjunction with thecounter electrode 240 of FIG. 2, the electrode of the microwellsprovides an electric field in the flow cell 106 of FIG. 2.

In an exemplary embodiment, the electrodes proximal to the well wallstructures and coupled to the terminal 230 are disposed at a base of thewell wall proximal to the passivation layer. Alternatively, theelectrode proximal to the well wall can be disposed within the well wallor on top of the well wall. In particular, the electrode can be disposedat positions between the top and the bottom of the well within the wellwall and in electrical contact with fluid within the well.

In particular, the electrode can be formed while manufacturing the wellwall structures that define the wells above the sensor pads. A devicesubstrate defines an array of sensors, such as the FETs illustrated inFIG. 1. Each of the sensors of the array of sensors includes anelectrode structure. The electrode structure can include a sensor padstructure exposed at the surface of the device substrate. As illustratedin FIG. 4, the pad structures 402 can be separated from each other byinsulative material 404. The pad structures 402 can form a portion of afloating gate structure, such as the sensor pad 120 of the floating gate118 illustrated in FIG. 1. In particular, the pad structures 402 can beformed of a conductive material, such as a conductive metal. Theinsulative material 404 can be silicon oxide, silicon nitride, siliconoxynitride, or a combination thereof.

As illustrated in FIG. 5, the pad structures 402 can be exposed throughthe insulative material 404 to form insulation structures 504. Forexample, resist can be applied over the insulative material 404, apattern can be implemented in the resist using lithography, portions ofthe insulating material 404 can be etched in accordance with thepattern, and any remaining resist can be stripped to expose surface ofthe pad structures 402. Etching can include wet etching or a plasmaetching. In particular example, etching includes plasma etching withfluorinated species, such as trifluoromethane, tetrafluoromethane,nitrogen fluoride, sulfur hexafluoride, or a combination thereof.

As illustrated in FIG. 6, a passivation layer 606 can be disposed overthe insulation structures 504 and the pad structures 402. For example,the passivation layer 606 can be deposited using atomic layerdeposition. The passivation layer 606 can also have insulativeproperties. In particular, layers of aluminum oxide, tantalum oxide, orcombinations thereof can be deposited using atomic layer deposition. Ina particular example, a three layer structure can be deposited includinga first layer of aluminum oxide, a second layer of tantalum oxide, and athird layer of aluminum oxide. The passivation layer 606 can have athickness in a range of 5 nm to 100 nm, such as 10 nm to 70 nm, such as15 nm to 65 nm, or even 20 nm to 50 nm. Alternatively, the insulationstructures 504 and the pad structures 402 can remain free of apassivation layer.

In an embodiment in which the electrode layer is deposited adjacent tothe passivation layer 606, an electrode layer 708 can be deposited overthe passivation layer 606, as illustrated in FIG. 7. In an example, theelectrode layer 708 can be deposited using techniques such assputtering. For example, the electrode layer can be formed of a materialsuch as gold, silver, platinum, aluminum, copper, or a combinationthereof. In an example, the electrode layer has a thickness in a rangeof 50 nm to 500 nm, such as a range of 80 nm to 400 nm, a range of 100nm to 300 nm, or even a range of 100 nm to 200 nm.

As illustrated in FIG. 8, the well wall structures can be formed of amonolithic insulative layer. For example, the layer 810 can be formedover the electrode layer 708. Alternatively, wells can be formed througha structure including more than one layer. In particular, the layer 810can be formed of silicon dioxide, silicon nitride, silicon oxynitride,tetra orthosilicate (TEOS), other insulative materials, or a combinationthereof. In particular, such materials can be deposited, for example,using chemical vapor deposition or other deposition techniques.Alternatively, the layer 810 can be formed of an insulative polymericmaterial, such as through spin coating or other coating techniques.

To form the well structures, openings can be formed through the layer810 to expose the electrode layer 708 above the pad structures 402. Forexample, as illustrated in FIG. 9, a photoresist can be used to patternlarge openings providing access to the layer 810. For example, thephotoresist, using lithography, can be used to form small blockingstructures 912 above the insulation structures 504.

Using an etch process, the openings can be formed to expose theelectrode layer 708 above the pad structures 402, as illustrated in FIG.10. For example, a fluorinated etch with endpoint detection can be used.Such a plasma etch technique can utilize a fluorinated species, such asthose described above, to form a fluorinated plasma that etches thematerial of the layer 810.

Following stripping of the photoresist and wet etch removal of a portionof the electrode layer 708 that extends over the pad structures 402, anelectrode layer 1114 is formed underneath the well wall structure 810and above the passivation layer 606 on top of the insulation structures504. In particular, the electrode layer 708 can be wet etched using anacid solution including phosphoric acid, acetic acid, nitric acid, or acombination thereof to form the electrode layer 1114. As such, theelectrophoresis electrode layer 1114 is exposed in wells of the array.In the case of a polymer insulative layer in place of layer 810, thepolymer layer can be etch in conjunction with the electrode layer 708,bypassing one or more steps of the illustrated process.

The electrode layer 114 is physically separate from the electrodestructure including the pad structure 402 and can electrically isolatedfrom the pad structure except when a conductive fluid is present in thewell. Over the pad structures 402, the passivation layer 606 is exposed.As discussed above, the electrode layer 1114 can be coupled with aterminal 230 of FIG. 2 and a charge differential can be induced betweenthe top of a flow channel and the interior of the wells.

In another embodiment, the electrode can be formed within the well wallstructure between the top and bottom of the well. Returning to FIG. 6, apassivation layer 606 is disposed over the pad structures 402 and theinsulation structures 504. As illustrated in FIG. 12, a first insulationlayer 1216 can be deposited over the passivation layer 606. For example,the insulation layer 1216 can be deposited using chemical vapordeposition and can be formed of materials, such as silicon dioxide,silicon nitride, silicon oxynitride, tetra orthosilicate (TEOS), or acombination thereof. Alternative, a polymeric insulative material can beused.

As illustrated in FIG. 13, an electrode layer 1318 can be deposited overthe first insulation layer 1216. The electrode layer 1318 can bedeposited using techniques, such as sputtering, and can be formed ofmaterials such as those described above having a range of thickness asdescribed above.

A second insulation layer 1420 can be deposited over the electrode layer1318, as illustrated in FIG. 14. For example, the second insulatinglayer 1420 can be deposited using chemical vapor deposition and can beformed of a material such as silicon dioxide, silicon nitride, siliconoxynitride, tetra orthosilicate (TEOS), or combination thereof.Alternatively, a polymeric insulative material can be used.

As illustrated in FIG. 15, photoresist structures 1522 can be formed,such as through lithographic techniques, above the insulation structures504. The photoresist structures 1512 can provide a large opening forforming wells within the well wall structure.

For example, as illustrated at FIG. 16, the second insulation layer 1420can be etched to the electrode layer 1318. For example, a fluorinatedetch technique with endpoint detection can be utilized. In particular, aplasma etch utilizing trifluoromethane, tetrafluoromethane, nitrogenfluoride, sulfur hexafluoride, or a combination thereof can be used toetch the second insulation layer 1420.

As illustrated in FIG. 17, the electrode layer 1318 can be etched toform the electrode layer 1718. In an example, the electrode layer 1318can be wet etched. In another example, the electrode layer 1318 can beplasma etched. For example, a chlorinated etch can be used to etchthrough the electrode layer. In particular, some overreach may bepermitted, etching beyond the electrode layer 1318 into a portion of thefirst insulation layer 1216.

As illustrated in FIG. 18, the first insulation layer 1216 can be etchedto expose the passivation layer 606 over the pad structures 402 or thepad structures 402 itself. For example, a fluorinated etch with endpointdetection can be utilized to etch the first insulating layer 1216. Theresulting structure includes electrode layer 1718 disposed within thewell wall structure 1822 between the top and bottom of the well 1824 andin electrical contact with fluid within the well 1824.

In an alternative embodiment, an electrode layer can be deposited on topof a well wall structure. For example, as illustrated in FIG. 19, anelectrode 1924 can be disposed atop a well wall structure 1922. Apassivation layer 606 can be positioned over the pad structures 402 andthe insulation structures 504. The passivation layer 606 can be exposedthrough the well wall structure 1922. In each of the above embodiments,a buffer layer can be dispensed over the well wall structures.Optionally, a buffer layer or an additional insulating layer 1926 can bedisposed over the electrode 1924.

In a further exemplary embodiment illustrated in FIG. 20, a structure2002 is provided. The structure 2002 can form the basis of a testdevice. In an alternative example, the structure 2002 can include anarray of sensors. Each of the sensors can include a gate oxide overwhich a floating gate and sensor pad can be disposed. As illustrated inFIG. 21, a metal layer 2104 can be deposited over the structure 2002. Inan example, the metal layer 2104 has a thickness in a range of 0.5 μm to1.0 μm. The metal layer 2104 can be deposited using a techniquedescribed above and can include a metal selected from those identifiedabove.

As illustrated in FIG. 22, a passivation layer 2206 can be depositedover the metal layer 2104. In a particular example, the passivationlayer 2206 is deposited using atomic layer deposition. In an example,the passivation layer 2206 can include a layer of aluminum oxide havinga thickness of approximately 20 nm deposited over the metal layer 2104,and can include a further layer of tantalum oxide having a thickness ofapproximately 40 nm deposited over the aluminum oxide.

As illustrated in FIG. 23, an electrode layer 2308 can be deposited overthe passivation layer 2206. In particular, the electrode layer 2308 canbe formed of a conductive material, such as a metal, deposited using amethod described above. In an example, the electrode layer 2308 has athickness in a range of 0.2 μm to 0.5 μm.

As illustrated in FIG. 24, an insulation layer 2410 can be depositedover the electrode layer 2308. In an example, the insulation layer 2410can be formed of an oxide, nitride, or combination thereof of silicon.Alternative, a polymeric insulative material can be used.

As illustrated in FIG. 25, a patterned photoresist layer 2512 can bedeposited over the insulation layer 2410. The insulation layer 2410 canbe etched, for example, using a fluorinated plasma etch to form a well2612. Using a further etch process, such as a wet etch process, theelectrode layer 2308 can be etched to expose the passivation layer 2206,as illustrated in FIG. 27.

Starting with FIG. 22, a bond pad portion of the device can be formed.For example, as illustrated in FIG. 28, a patterned photoresist layer2808 can be applied over the passivation layer 2206. As illustrated inFIG. 29, the passivation layer 2206 can be etched to expose theunderlying metal layer 2104. An electrode layer 3008 can be depositedover the patterned passivation layer 2206 and the exposed metal layer2104, as illustrated in FIG. 30. Optionally, the electrode layer 3008can be formed in a same deposition as the layer 2308 of FIG. 23. Theelectrode layer 3008 and the layer 2308 can be connected. Alternatively,the electrode layer 3008 and the layer 2308 can be electricallyisolated.

As illustrated in FIG. 31, an insulation layer 3110 can be depositedover the metal layer 3008. As illustrated in FIG. 32, a patternedphotoresist layer 3212 is deposited over the insulation layer 3110. Theinsulation layer 3110 can be etched to expose the electrode layer 3008.An interconnect with the device can be formed by depositing a conductivematerial into the opening 3314.

In an additional embodiment, the electrode layer can be formed within awell structure of the well wall. Starting with FIG. 22, a metal layer2104 is deposited over a sensor structure 2002. A passivation layer 2206is deposited over the metal layer 2104. As illustrated in FIG. 34, aninsulation layer 3408 is deposited over the passivation layer 2206. Anelectrode layer 3510 can be deposited over the insulation layer 3408, asillustrated in FIG. 35. An additional insulation layer 3612 can bedeposited over the electrode layer 3510, as illustrated in FIG. 36.

The layers can be patterned. For example, as illustrated in FIG. 37, apatterned photoresist layer 3714 can be this deposited over theinsulation layer 3612, the electrode layer 3510, and the insulationlayer 3408. As illustrated in FIG. 38, the insulation layer 3612 can beetched to provide an initial well 3811. For example, etching can includeplasma etching, such as a fluorinated plasma etch. The electrode layer3510 can be etched, for example, using a wet patch process, asillustrated in FIG. 39. Further, the insulation layer 3408 can beetched, for example, using a plasma etch process, such as a fluorinatedplasma etch. As a result, the passivation layer 2206 disposed over themetal layer 2104 is exposed through the well 3811, as illustrated inFIG. 40.

Bond pads can be formed in a different portion of the device. Forexample, starting with FIG. 34, a passivation layer 2206 is depositedover a metal layer 2104, and an insulation layer 2408 is deposited overthe passivation layer 2206. As illustrated in FIG. 41, a patterned layer4110 of photoresist can be applied over the insulation layer 3408. Thelayer 3408 can be patterned or etched in accordance with the pattern ofthe layer 4110. For example, as illustrated in FIG. 42, the layer 3408can be etched using a plasma, such as a fluorinated plasma.

As illustrated in FIG. 43, an electrode layer 4312 is deposited over theinsulation layer 3408 and passivation layer 2206. An additionalinsulation layer 4414 can be deposited over the electrode layer 4312. Asillustrated in FIG. 45, a patterned photoresist layer 4518 can beapplied over the layer 4414. After etching, a well 4620 is formed in thelayer 4414, exposing the electrode layer 4312, as illustrated in FIG.46. A conductive material can be deposited into the well 4620 to form aninterconnect.

In relation to FIGS. 20-46, techniques and materials utilized inrelation to FIGS. 4-19 can be utilized. Further, while a layer, such asa metal layer may be illustrated as a single continuous layer, such alayer may be patterned and isolated from other metal layers outside ofthe illustrated cross-section. In addition, while not illustrated,electrical interconnects can be formed by filling voids over bond padswith conductive material. Further, while the methods for forming bondpads are provided in relation to FIGS. 20-46, similar methods can beutilized for bond pad manufacturing for those processes illustrated inFIGS. 4-19. In addition, the shape of the side walls of the illustratedwells can have a vertical orientation, an outward sloping orientation,an inward sloping orientation, or a combination thereof.

The above structures can be used to load particles into wells of adevice. As illustrated in FIG. 47, a particle suspension can be providedto a flow cell of an apparatus, as illustrated at 4702. For example, asillustrated in FIG. 2, a flow cell is defined above an array of wellsand sensors. The particle suspension can include particles in a fluid.The fluid can be electrically conductive. Alternatively, the fluid canbe non-conductive. In an example, the particles are charged particles.In a particular example, the particles are conjugated withpolynucleotides.

With the particle suspension in the flow cell, a voltage difference canbe applied between a counter electrode and the electrophoresiselectrode, as illustrated at 4704, providing an electric field. Thevoltage difference can be a DC voltage difference or can be an ACvoltage difference, such as a DC biased AC voltage difference. Thevoltage difference can be facilitated by activating a voltage sourceelectrically connected to the counter electrode and the electrophoresiselectrode.

When the apparatus is configured for use in genetic detection methods,such as sequencing, a nucleotide solution can be provided through theflow cell, as illustrated at 4706. For example, the nucleotide solutioncan include a single type of nucleotide in solution. In another example,the nucleotide solution can include more than one nucleotide type. Aresponse, such as an ionic response, to the addition of the nucleotidesolution can be measured by the sensors, as illustrated at 4708. Forexample, the sensors can be ion sensitive field effect transistors.

In a first aspect, an apparatus includes a device substrate including anarray of sensors. Each sensor of the array of sensors includes anelectrode structure disposed at a surface of the device substrate. Theapparatus further includes a well structure overlying the surface of thedevice substrate and defining an array of wells at least partiallycorresponding with the array of sensors. The well structure includes anelectrode layer and an insulative layer.

In an example of the first aspect, a well of the array of wells is toprovide fluid access to a sensor of the array of sensors.

In another example of the first aspect and the above examples, a well ofthe array of wells exposes the electrode structure of a sensor of thearray of sensors.

In a further example of the first aspect and the above examples, theelectrode layer is exposed in a plurality of wells of the array ofwells.

In additional example of the first aspect and the above examples, theelectrode layer is electrically connected to a plurality of wells of thearray of wells.

In another example of the first aspect and the above examples, theapparatus further includes an electrical interconnect separate from thearray of wells providing electrical access to the electrode layer.

In a further example of the first aspect and the above examples, theinsulative layer is disposed below the electrode layer and wherein asecond insulative layer is disposed above the electrode layer, the arrayof wells defined through the insulative layer, the electrode layer, andthe second insulative layer.

In an additional example of the first aspect and the above examples, asensor of the sensor array is an ion sensitive field effect transistor.

In another example of the first aspect and the above examples, theelectrode structure includes a floating electrode.

In an additional example of the first aspect and the above examples, theelectrode structure is electrically isolated from the electrode layerexcept through fluid within the array of wells. In a further example ofthe first aspect and the above examples, the apparatus further includesa cover defining a flow cell over the wall structure, the flow cell tocontain a fluid. In another example of the first aspect and the aboveexamples, the apparatus further includes a counter electrode and avoltage source electrically coupled to the electrode layer and thecounter electrode.

In a second aspect, a method of forming a sensor apparatus includesapplying a first insulative layer over a device substrate. The devicesubstrate includes an array of sensors. Each sensor of the array ofsensors includes an electrode structure at the surface of the devicesubstrate. The method further includes applying an electrode layer overthe first insulative layer, applying a second insulative layer over theelectrode layer, and forming an array of wells in the first insulativelayer, the electrode layer and the second insulative layer. The array ofwells substantially corresponds with the electrodes of the sensors ofthe array of sensors.

In an example of the second aspect, forming the array of wells includesetching the second insulative layer proximal to the electrode layer,etching the electrode layer proximal to the first insulative layer, andetching the first insulative layer to expose the electrode structure.

In another example of the second aspect and the above examples, themethod further includes forming an interconnect in contact with theelectrode layer.

In a further example of the second aspect and the above examples,forming the array of wells includes exposing electrode structures of thearray of sensors.

In an additional example of the second aspect and the above examples,the electrode layer is electrically connected to a plurality of wells ofthe array of wells.

In another example of the second aspect and the above examples, a sensorof the sensor array is an ion sensitive field effect transistor.

In a further example of the second aspect and the above examples, theelectrode structure includes a floating electrode.

In an additional example of the second aspect and the above examples,the electrode structure is electrically isolated from the electrodelayer except through fluid within the array of wells.

In another example of the second aspect and the above examples, themethod further includes applying a cover defining a flow cell over thewall structure.

In a further example of the second aspect and the above examples, themethod further includes providing a counter electrode and a voltagesource electrically coupled to the electrode layer and the counterelectrode.

In a third aspect, a method of loading particles includes providing aparticle suspension into a flow cell of an apparatus. The particlesuspension includes a plurality of particles in a fluid. The apparatusincludes a device substrate including an array of sensors. Each sensorof the array of sensors includes an electrode structure disposed at asurface of the device substrate. The apparatus further includes a wellstructure overlying the surface of the device substrate and defining anarray of wells at least partially corresponding with the array ofsensors. The well structure includes an electrode layer and aninsulative layer. The apparatus further includes a counter electrode anda voltage source electrically coupled to the electrode layer and thecounter electrode. The method further includes activating the voltagesource to provide a voltage difference between the electrode layer andthe counter electrode, whereby the particles are motivated into wells ofthe array of wells.

In an example of the third aspect, activating the voltage sourceincludes applying a DC voltage difference between the electrode layerand the counter electrode. In another example of the third aspect andthe above examples, activating the voltage source includes applying anAC voltage difference between the electrode layer and the counterelectrode. In an example, the AC voltage difference is a DC biased ACvoltage difference.

In a further example of the third aspect and the above examples, theplurality of particles includes nucleic acid-containing particles.

In an additional example of the third aspect and the above examples, themethod further includes flowing a nucleotide solution through the flowcell and measuring an ionic response using the electrode structure.

Note that not all of the activities described above in the generaldescription or the examples are required, that a portion of a specificactivity may not be required, and that one or more further activitiesmay be performed in addition to those described. Still further, theorder in which activities are listed are not necessarily the order inwhich they are performed.

In the foregoing specification, the concepts have been described withreference to specific embodiments. However, one of ordinary skill in theart appreciates that various modifications and changes can be madewithout departing from the scope of the invention as set forth in theclaims below. Accordingly, the specification and figures are to beregarded in an illustrative rather than a restrictive sense, and allsuch modifications are intended to be included within the scope ofinvention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of features is notnecessarily limited only to those features but may include otherfeatures not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive-or and not to an exclusive-or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the use of “a” or “an” are employed to describe elements andcomponents described herein. This is done merely for convenience and togive a general sense of the scope of the invention. This descriptionshould be read to include one or at least one and the singular alsoincludes the plural unless it is obvious that it is meant otherwise.

Benefits, other advantages, and solutions to problems have beendescribed above with regard to specific embodiments. However, thebenefits, advantages, solutions to problems, and any feature(s) that maycause any benefit, advantage, or solution to occur or become morepronounced are not to be construed as a critical, required, or essentialfeature of any or all the claims.

After reading the specification, skilled artisans will appreciate thatcertain features are, for clarity, described herein in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, references to valuesstated in ranges include each and every value within that range.

What is claimed is:
 1. A method of forming a sensor apparatus, themethod comprising: applying a first insulative layer over a devicesubstrate, the device substrate including an array of sensors, eachsensor of the array of sensors including an electrode structure at thesurface of the device substrate; applying an electrode layer over thefirst insulative layer; applying a second insulative layer over theelectrode layer; and forming an array of wells in the first insulativelayer, the electrode layer and the second insulative layer, the array ofwells substantially corresponding with the electrodes of the sensors ofthe array of sensors.
 2. The method of claim 1, wherein forming thearray of wells comprises: etching the second insulative layer proximalto the electrode layer; etching the electrode layer proximal to thefirst insulative layer; and etching the first insulative layer to exposethe electrode structure.
 3. The method of claim 1, further comprisingforming an interconnect in contact with the electrode layer.
 4. Themethod of claim 1, wherein forming the array of wells includes exposingelectrode structures of the array of sensors.
 5. The method of claim 1,wherein the electrode layer is electrically connected to a plurality ofwells of the array of wells.
 6. The method of claim 1, wherein a sensorof the sensor array is an ion sensitive field effect transistor.
 7. Themethod of claim 1, wherein the electrode structure includes a floatingelectrode.
 8. The method of claim 1, wherein the electrode structure iselectrically isolated from the electrode layer except through fluidwithin the array of wells.